PROJECT SUMMARY/ABSTRACT Clostridium difficile is the main cause of antibiotic-associated diarrhea. Since 2003, the incidence and severity of C. difficile infection (CDI) has risen in the U.S. and globally. In 2013 the CDC designated C. difficile as an Urgent Threat, which caused ~29,000 deaths from ~453,000 cases in 2011. These trends were related to the emergence of epidemic strains, particularly epidemic 027. Epidemic 027 causes about a third of CDI in the U.S. Metronidazole is the most commonly prescribed drug for CDI, but after 30 years of use, it is now associated with poorer treatment outcomes. Reasons for the now poorer outcomes for metronidazole therapy is unclear. A subset of epidemic 027 strains show decreased susceptibility to metronidazole, when tested in the cofactor heme. Similarly, other dominant epidemic strains associated with CDI in the United States have evolved metronidazole resistance that is expressed in heme. While resistance has evolved in clinical strains, it is unclear how this impacts the ability to treat CDI with metronidazole. This study addresses this medically important question, by investigating how metronidazole resistance affects treatment outcomes. Firstly, a biobank of patient stools is examined for metronidazole-resistant strains, heme levels and patient metadata for clinical outcomes. This plan is complemented by experimental CDI models, namely the in vitro human gut and hamster models of CDI that are clinically reflective. Also addressed is the question of whether the low concentrations of metronidazole in the colon of patients is inadequate to treat infections with metronidazole- resistant strains. The genetic mechanisms adopted by C. difficile to display resistance is still unclear. Hence, this study elucidates the genetic basis for the evolution of metronidazole resistance in C. difficile, using a combination of cutting-edge genetic algorithms to identify gene changes in the genomic data sets for resistant isolates when compared to sensitive isolates and by molecular genetics to recapitulate metronidazole resistance in nave strains. Public health. The successful completion of this study will immediately impact healthcare practices and the guidelines for CDI management. This could save lives by improving prescribing practices and guideline adherence.